亚硝酸盐胁迫下植物乳杆菌WU14亚硝酸盐还原酶的食品级高效诱导表达及其酶学性质研究  

Food-Grade Induced Expression and Enzymatic Properties of Nitrite Reductase from Lactobacillus plantarum WU14 Under Nitrite Stress

作　　者：应碧 昌晓宇[1] 刘志文[1] 周通[1] 陈瑶[1] 钟平安[1] 徐波[1] 

机构地区：[1]江西农业大学生物科学与工程学院,南昌330045

出　　处：《中国农业科学》2015年第7期1415-1427,共13页Scientia Agricultura Sinica

基　　金：国家自然科学基金(31360372);江西省教育厅科研基金(GJJ11407);南昌市科技局科研项目(洪科发计字(2011)158-52);江西省大学生创新创业训练计划项目(201410410041)

摘　　要：【目的】研究亚硝酸盐胁迫下植物乳杆菌(Lactobacillus plantarum)WU14亚硝酸盐还原酶(nitrite reductase,Nir S)的作用机制,为发酵食品中应用乳酸菌纯种培养技术降解亚硝酸盐奠定基础。【方法】在37℃培养条件下,测定含0.02%—0.16%Na NO2的MRS培养液中L.plantarum WU14 24 h的生长密度、p H及Na NO2降解量。通过PCR扩增和TA克隆得到Nir S,并克隆到乳酸乳球菌食品级细胞内高效诱导表达载体p RNA48中,获得重组菌L.lactis NZ9000/p RNA48-Nir S。重组菌经30 ng·m L-1 nisin诱导后,经SDS-PAGE和盐酸萘乙二胺法分析目的蛋白的表达情况及Nir S酶活。通过生物信息学软件预测分析Nir S编码的蛋白质二三级结构、跨膜结构及疏水性。【结果】L.plantarum WU14能够在Na NO2浓度小于0.12%的MRS培养基中生长并降解一部分Na NO2,在0.10%Na NO2培养液中发酵24 h后降解量达到最大,为56.34μg·m L-1,Nir S酶活达2 347.5 U·m L-1。Nir S编码的蛋白质是一种以α-螺旋和无规则卷曲为主,不存在信号肽和跨膜结构的亲水蛋白。Nir S可在L.lactis NZ9000中高效表达,该重组菌能够在Na NO2浓度低于0.10%的GM17培养基中生长并降解一部分Na NO2,在含0.04%Na NO2的培养液中亚硝酸盐降解量达到最大,为22.21 mg·m L-1,Nir S酶活为925.41 U·m L-1。【结论】L.plantarum WU14的Nir S能够降解高浓度的亚硝酸盐,并且经食品级异源表达的Nir S具有较高的酶活力。本研究为探索研究亚硝酸盐降解的机理,建立发酵食品中亚硝酸盐降解的可控发酵体系提供了参考。[Objective]The study aimed to explore the mechanism of nitrite reductase from Lactobacillus plantarum WU14 under nitrite stress so that lay a foundation for pure culture technology of lactic acid bacteria in fermented food. [Method] Growth density, pH and nitrite degradation quantity of L. plantarum WU14 were determined when the liquid medium contained sodium nitrite ranged from 0.02% to 0.16% under the condition of 37℃. The recombination strain Lactococcus lactic NZ9000/pRNA48-NirS was constructed followed the putative nitrite reductase gene from L. plantarum WU14 was amplified by PCR and then cloned into the food-grade cytoplasmic inducible expression vector pRNA48 of L. lactic NZ9000. After induced with 30 ng·mL^-1 nisin, the expressed target protein and the enzyme activity of nitrite reductase of the recombinant strains were analyzed by SDS-PAGE and Naphthyl ethylenediamine dihydrochloride spectrophotometric method. Using the bioinformatics software, the high level protein structure, membrane structure and hydrophobicity of nitrite reductase gene were predicted and analyzed. [Result]L. plantarum WU14 could routinely grow in MRS medium containing less than 0.12% nitrite, along with degradation of nitrite. After the strain L. plantarum WU14 was cultured for 24 hours in the liquid medium containing 0.10% sodium nitrite, the nitrite reductase activity of L. plantarum WU14 was 2 347.5 U·mL^-1, and the degradation quantity was 56.34 μg·mL^-1 according to the analysis of its nitrite degradation ability. the NirS gene could express in the recombinant strain. Nitrite reductase gene encodes a kind of hydrophilic protein containing alpha helix and random coil, no signal peptide and transmembrane structure. The recombination strain could routinely grow in GM17 medium containing less than 0.10% nitrite, meanwhile, its enzyme activity reached 925.41 U·mL^-1 and the degradation quantity reached 22.21 mg·mL^-1 after 24 h fermentation in the 0.04% nitrite concentration medium. [Conclusion]Nitrite reductase from L. pla

关 键 词：植物乳杆菌WU14 亚硝酸盐胁迫 亚硝酸盐还原酶 食品级高效诱导表达 

分 类 号：TS201.3[轻工技术与工程—食品科学]